Antibodies specific for peptide corresponding to CD44 exon 6, and use of these antibodies for diagnosis of tumors

ABSTRACT

There is marked over-expression of multiple spliced variants of the CD44 gene in tumor compared to counterpart normal tissue. This observation forms the basis of a method of diagnosing neoplasia by analysis of a sample of body tissue or body fluid or waste product. A new exon 6 of 129 bp has been located and sequenced. Antibodies specific to the exon have been prepared and are claimed as new compounds suitable for use in the detection of CD44 proteins and for the in vivo imaging and therapy of tumors.

BACKGROUND

The present invention is concerned with using expression of the CD44gene or part of the CD44 gene to investigate neoplasia. Suchinvestigation includes taking a tissue, body fluid or other sample froma patient to perform diagnosis, to give a prognosis or to evaluatetherapy that is already being carried out. In particular, the inventionprovides a simple method for carrying out routine screening forneoplasia using body fluid samples or other samples which can beobtained non-invasively.

The usual way to diagnose a tumour at present is by looking at cells orthin slices of tissue down a microscope, a method which is often veryeffective but has some important limitations. With a small sample,diagnosis can be very difficult and often a large number of cells willnot be available, or it is not desirable or possible to obtain a largesample from the patient. In as many as 50% of cases a reliable diagnosiscannot be given; it may be that there is no positive evidence ofcarcinoma but also no certainty that the patient is actually free fromcarcinoma. More invasive investigation is then required to establish adiagnosis.

Judgment of prognosis also relies on the appearance of cells when viewedunder a microscope. Generally, the more bizarre-looking the cells in aprimary tumour, the more likely they are to metastasise later on but thecorrelation is by no means absolute. It would clearly be an advantage tobe able to predict more accurately whether or not metastasis is likelyto occur in order to judge what will be the most effective treatment.

The human CD44 gene codes for a family of variably glycosylated cellsurface proteins of different sizes, the numerous functions of which arenot yet fully established, but which share epitopes recognised by theCD44 monoclonal antibody (mAb). It is known to consist of a standardportion which is expressed in haemopoietic cells and many other celltypes and into which the products of additional exons may be spliced invarious combinations to produce different proteins. This is a wellrecognised mechanism in eukaryotes for producing several oftenfunctionally unrelated proteins from the same gene, and is known asalternative splicing.

Two common CD44 isoforms have so far been purified and characterised(Stamenkovic et al. 1989), namely i) a 90 kD form consisting of acentral 37 kD core which is heavily glycosylated and ii) a 180 kD formwhich has 135 extra amino acids inserted into the proximalextra-membrane domain and is even more heavily glycosylated.Immuno-cytochemical and immuno-precipitation studies have shown thatboth are widely distributed in many different cells and tissues. Theformer is known as the haemopoietic or standard form which is present oncirculating leukocytes, bone marrow cells and numerous other cell types.The other, known as the epithelial variant, is detectable on severalepithelial cell types. Both are believed to function as receptorsmediating homotypic and heterotypic adhesive interactions, attachingcells to each other or to adjacent extracellular scaffolding.

Some time ago, some of the CD44 epitopes recognised by the mAb Hermes-3were identified as constituting the peripheral lymph node receptorenabling circulating lymphocytes to recognise and traffic throughperipheral lymph nodes. Further mAbs to this antigen later becameavailable and Stamenkovic et al. (1989) used one of them to clone a cDNAsequence coding for the standard form of the molecule from an expressionlibrary in COS cells. They additionally found, by Northern blotting,that this gene was expressed not only by lymphoid cells, but also by avariety of carcinoma cell lines and a representative sample of solidcarcinomas, amongst which two colonic carcinomas appeared to expressmore than normal colonic epithelium.

Birch and colleagues (1991) reported that melanoma cell clones whichstrongly expressed the 80-90 kD form of the CD44 antigen, recognised bythe Hermes-3 antibody, were substantially more metastatic in nude micethan clones which expressed it weakly. Sy et al. (1991) described amoderate increase in metastatic capability of human lymphoma cells innude mice, after the cells were transfected with the standard CD44 gene,but not after transfection with a construct coding for the epithelialvariant. Gunthert et al. (1991) obtained results indicating that avariant form of the lymphocyte homing receptor, recognised by a newantibody raised to the rat CD44 antigen, is required for metastaticbehaviour of rat pancreatic adenocarcinoma cells. Using this antibodythey cloned a cDNA sequence corresponding to the variant form of CD44and found that it contained previously unidentified exons. Transfectionof a non-metastatic clone from the same cell line with a constructdesigned to over-express this cDNA sequence unique to the metastaticcounterpart, appeared to induce metastatic behaviour (Gunthert et al,1991).

In view of these findings it became of interest to know whether othercultured metastatic and non-metastatic human tumour cell lines, ofvarious histogenetic origins, expressed CD44 produces differentially.The expression of genes in cells or tissues can be studied mostefficiently and sensitively by making cDNA from cellular messenger RNAand amplifying regions of interest with the polymerase chain reaction,using specific oligonucleotide primers chosen to anneal preferentiallyto portions of the cDNA corresponding to the gene products. However,subsequent work by Hofmann et al. (1991) and the present applicantsusing this approach provided results which showed that CD44 expressiondid not regularly and reliably correlate with the metastatic capabilityor even tumour forming ability of these cultured cell lines in nudemice. At about this time, three separate groups (Hofmann et al, 1991,Stamenkovic et al, 1991 and Jackson et al, 1992) published sequence dataon further splice variants they had found being expressed by this genein various human cell lines.

THE INVENTION

The present invention results from a surprising discovery resulting fromstudies examining the expression of various parts of the CD44 gene infresh tissue and body fluid samples from patients with tumours of thebreast and colon and from their metastases. The results indicate sharpand clear differences in CD44 expression between tissues from i)metastatic (malignant) tumours, ii) non-metastatic locally invasivetumours and benign tumours and iii) normal tissue. The distinctionbetween groups i) and ii) is important for judgment of therapy and thatbetween groups ii) and iii) is important for early diagnosis andscreening.

Part of this invention forms the subject of our International patentapplication PCT/GB93/01520 filed 20 Jul. 1993, which provides in oneaspect a method of diagnosis of neoplasia, which method comprisesanalysing the expression of the CD44 gene in a sample.

In a particular embodiment, that application provides a method ofassaying a sample for products of the CD44 gene or part thereof whichmethod comprises making cDNA from messenger RNA (mRNA) in the sample,amplifying portions of the complementary DNA (cDNA) corresponding to theCD44 gene or part thereof and detecting the amplified cDNA,characterised in that the amplified cDNA is used in diagnosis ofneoplasia.

The diagnosis of neoplasia may refer to the initial detection ofneoplastic tissue or it may be the step of distinguishing betweenmetastatic and non-metastatic tumours. References to the term"diagnosis" as used herein are to be understood accordingly.

The method is particularly applicable to the diagnosis of solid tumoursparticularly malignant tumours e.g. carcinomas. The sample on which theassay is performed is preferably of body tissue or body fluid; and notof cells cultured in vitro. The sample may be a small piece of tissue ora fine needle aspirate (FNA) of cells from a solid tumour.Alternatively, it may be a sample of blood or urine or another bodyfluid, a cervical scraping or a non-invasively obtained sample such assputum, urine or stool.

The cDNA may be detected by use of one or more labelled specificoligonucleotide probes, the probes being chosen so as to be capable ofannealing to part of the amplified cDNA sequence. Alternatively,labelled oligonucleotide primers and/or labelled mononucleotides couldbe used. There are a number of suitable detectable labels which can beemployed, including radiolabels.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference is directed to the accompanying drawings, in which:

FIGS. 1A, 1B, 1C, 2A, 2B, 2C, 3, 4, 5A and 5B are autoradiographsshowing the results of various experiments reported below,

FIG. 6 is a map of the CD44 gene showing exons, probes and primers. Thenumbering of the exons corresponds to that used by G. R. Screaton et al.1992),

FIG. 7 (SEQ ID NO:1) is the nucleic acid sequence of Exon 6 (shown inFIG. 6), the corresponding amino acid sequence being also shown (SEQ IDNO:2), and

FIG. 8 is a set of autoradiographs showing the results of anotherexperiment.

FIG. 9 is the DNA sequence of HIV2(gp32)-CD44 exon 6 fusiongene.

FIG. 10 is the protein sequence of HIV2(gp32)-CD44 exon 6 fusionantigen.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 6 is a map of the CD44 gene showing exons 6 to 14. The basic orstandard protein can theoretically be modified by the insertion oftranscripts from any, some, or all of these 9 extra exons. Exon 6 wasunknown at the priority date of this patent application, and constitutesa further aspect of the invention. Exon 6 is over-expressed in tumoursbut not in normal tissues, and is located in the vicinity of exons 7 to9. The sequence of exon 6 is given in FIG. 7. It contains 129 base pairsand is flanked on the 5'-side by the standard CD44 sequence, and on the3'-side usually by exon 7.

In contrast to Exons 9 to 11, the products of Exon 6 (thenewly-sequenced Exon) are only barely detectable in samples of normaltissues. This suggests that Exon 6 will be of particular value in thediagnosis of neoplasia.

In another aspect, that application provides as new compounds, thenucleic acid sequence of Exon 6 as shown in FIG. 7, characteristicfragments thereof, sequences which are degenerated and/or representallele variations, the homologous nucleic acid sequences, and probes,primers and other reagents capable of hybridising with the sequences orhomologues. These compounds and reagents will all be useful in themethod described above.

In accordance with the present invention the peptide sequencecorresponding to CD44 exon 6 as shown in FIG. 7, its allele variationsand secondary modifications thereof and characteristic fragments thereofcan be used the generate antibodies useful for the in vitro and in vivodiagnosis. Said antibodies are specific to the peptide corresponding toCD44 exon 6 as shown in FIG. 7, its allele variations and secondarymodifications thereof and characteristic fragments thereof i.e. theseantibodies bind to this peptide and possess a low cross-reactivitytowards other related CD44 proteins and other proteins. Said antibodiesmay be monoclonal or polyclonal. The antibodies may be generated byusing the entire peptide sequence corresponding to CD44 exon 6 as shownin FIG. 7 as an antigen or by using short peptides preferably of aminimum length of six amino acids encoding portions of the peptidesequence corresponding to CD44 exon 6, as antigens. The peptides used asantigens can be produced recombinantely or chemically by methods knownin the art. The peptide antigens according to the invention can forexample be synthesized according to Merryfield, JACS 85 (1964), 2146.For immunogen synthesis these peptides can be coupled to a carriermolecule for example keyhole limpet hemocyanin (KLH) or bovine serumalbumin (BSA). If a biotinylation is required this can for example becarried out according to PNAS U.S.A. 80 (1983), 4045.

For the expression of the peptide corresponding to CD44 exon 6 or itsallele variations in a procaryotic host it is prefered to prepare afusion gene of CD44 exon 6 or its allele variations with a gene whichpossess a high expression level in this host. For example a part of thegene encoding for the protein gp 32 of HIV 2 is suitable for E. coli.Thereby a fusion protein which possesses as a part the peptide sequenceaccording to Exon 6 or its allele variations is obtained. It is alsoprefered to increase the number of the peptide epitopes corresponding toCD44 exon 6 in such a fusion protein for example by duplicating the CD44exon 6 gene in the fusion gene.

Polyclonal antibodies directed against the peptide sequencecorresponding to CD44 exon 6, its allele variations and secondarymodifications thereof and characteristic fragments are prepared byinjection of suitable laboratory animal with an effective amount of apeptide or antigenic component, collecting serum from the animal, andisolating specific antibodies by any of the known immuno absorbenttechniques. Although the polyclonal antibodies produced by this methodare utilizable in any type of immunoassay, they are generally lessfavoured because of the potential heterogeneity.

The use of monoclonal antibodies in the in vitro diagnostic test isparticularly preferred because large quantities of antibodies all ofsimilar specificity may be produced. The preparation of hybridoma celllines for monoclonal antibody production is done by fusion of animmortal cell line and the antibody producing lymphocytes. This can bedone by techniques which are well known in the art (see for exampleHarlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold SpringHarbour Press 1988, Bessler et al. Immunobiol. 170 (1985), 239-244, Junget al., Angew. Chemie 97 (1985), 883 or Cianfriglia et al., HybridomaVol. 2, (1983), 451-457).

The following hybridoma cell lines which are producing monoclonalantibodies directed against the expression product of CD44 exon 6 asshown in FIG. 7 or a fragment thereof e. g. a characteristic epitopewere deposited on 16. Nov. 1993 under the Budapest Treaty at the DSM(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1b, D-3300 Braunschweig, Federal Republic of Germany:

MAK<CD44>M-1.1.12 DSM ACC2156

MAK<CD44>M-2.42.3 DSM ACC2157

MAK<CD44>M-4.3.16 DSN ACC2158

For MAK<CD44>M-1.1.12 a synthetic peptide corresponding to amino acids9-23, for MAK<CD44>M-2.42.3 a synthetic peptide corresponding to aminoacids 29-43 and for MAK<CD44>M-4.3.16 a synthetic peptide correspondingto amino acids 1-13 of the CD44 exon 6 peptide having the amino acidsequence shown in FIG. 7 was used. The antibody produced by the cellline MAK<CD44>M-1.1.12 shows a specificity to tumor tissue of lung,colon and bladder and for cells of the cell line ZR75-1 (human breastcarcinoma--ATCC CRL 1500) as detected by immunohistochemistry. Aspecific reaction means that a strong reaction is observed with thetumor tissue whereas normal tissue shows only a weak reaction. In thesame system the antibody produced by the cell line MAK<CD44>M-4.3.16shows specificity towards tumor tissue of colon and ZR75-1 cells.

The presence of the CD 44 protein or the peptide sequence according toCD44 exon 6 in a sample can be detected utilizing antibodies prepared asdiscribed above either monoclonal or polyclonal in virtually any type ofimmunoassay. A wide range of immunoassay techniques are available as canbe seen by reference to Harlow, et al. (Antibodies: A Laboratory Manual,Cold Spring Harbour Press 1988). This of course includes bothsingle-site and two-site, or "sandwich" of the non-competitive types, aswell as competitive binding assays. Sandwich assays are among the mostuseful and commonly used assays. A number of variations of the sandwichassay technique exist, and all are intended to be encompassed by thepresent invention. Examples for those assays are radio immunoassays,enzyme immunoassays or immunofluorescent assays such as FPIA orelectrochemilumineszent assays, immunoassays using direct labels such asdye particles (e.g. gold sol particles), homogeneous immunoassays suchas CEDIA or EMIT or turbidimetric and nephelometric methods such aslatex particle agglutination assays. It is possible to use twoantibodies according to the invention in a sandwich assay. In this casethese two antibodies must bind to different epitopes or sites of thepeptide sequence according to CD44 exon 6. These antibodies could forexample be prepared by using two different synthetic peptides asimmunogens corresponding to different characteristic fragments of thepeptide corresponding to CD44 exon 6. It is also possible to use onlyone antibody according to the invention in a sandwich assay. The otherantibody could be an antibody to the other peptides corresponding toother CD44 exons or to the standard form of CD44. Such antibodies areknown in the art.

It is possible to use for example urine, whole blood, cervical smears,stool, tissue for example biopsies, sputum or cells as sample. In mostcases the CD44 protein could be detected in its native form. Preferablythe CD44 protein is denatured prior to or during its detection becausesome of the antibodies according to the invention preferably bind toepitopes which are linear or which are hidden within the CD44 moleculein its native form. As a denaturation method any method known in the artsuch as treatment with detergent or chaotropic agents is suitable. Insome cases the adsorption of the CD44 molecule to a solid phase leads toa partial denaturation which is sufficient for the binding of theantibody.

Although CD44 proteins are expressed on most cell types it was foundthat with the use of the antibodies according to the invention adifferentiation between tumor tissue and normal tissue is possible inmost cases. The antibodies therefore could be used in cancer diagnosis.Preferably the antibodies could be used for the diagnosis for cancer oftissue of colon, bladder or lung. For example with the antibodyobtainable from the hybridoma cell line MAK<CD44>M-1.1.12 a strongreaction is observed in immunohistochemistry with colon, bladder or lungcarcinoma tissue wheras normal tissue of this origin gives only a weakreaction.

It is also possible to utilise the antibodies according to the inventionin immune complex analysis for example in a method according to Wong etal., Arch. Surg. 125 (1990), 187-191. Thereby the detection oftumor-associated immune complexes of CD44 protein or characteristicparts thereof and autoantibodies is possible.

The peptide antigens according to the invention can also be used as astandard compound in immunological tests for the quantitativedetermination of CD 44. The invention therefore in addition concerns theuse of the peptide antigens according to the invention as a standard inan immunological test for the determination of CD 44. In certain cases,for example in agglutination tests, it may be advantageous to bindseveral peptides according to the invention with the same or differentsequences to a carrier molecule. The peptides according to the inventioncan also be used as a binding partner for the antibody according to theinvention in a competitive immunoassay. In this case the peptides arelabeled or bound to a solid phase directly or indirectly via twospecific binding partners such as (strept)avidin/biotin by methods knownin the art.

Another aspect of the invention is a test kit containing at least oneantibody which is directed against the peptide corresponding to CD44exon 6 having the amino acid sequence as shown in FIG. 7, its allelevariations or secondary modifications thereof or characteristicfragments thereof among the other compounds which are necessary for theimmunoassay such as buffers, detergents, stabilizers, solid phases etc.If required the peptide antigens according to the invention as astandard could also be included.

In still another important aspect, this invention provides a means fortherapy and in vivo imaging of tumours. Agents useful for this can bemanufactured according to the state of the art. The data obtained fromstudies examining the expression of various parts of the CD44 gene insamples from patients with malignant diseases surprisingly show asignificant overexpression of exon 6 of the variable part of CD44. Therelative abundance of CD44 splice variants containing exon 6 inmalignant tumours as compared to normal tissue and the increased amountof CD44 proteins containing the peptide sequence encoded by exon 6 onthe surface of tumour cells as compared to normal tissue opens thepossibility to use the exon 6 encoded peptide sequence as a tumourspecific antigen for therapy, diagnosis both in vivo and in vitro, andin vivo imaging.

Preferably, monoclonal antibodies (Kohler and Milstein (1975), Nature156, 495-497) or their derivatives will be used for diagnostic andtherapeutic purposes. In this invention, monoclonal antibodies toepitopes encoded by exon 6 of CD44 are provided. Furthermore, data arepresented, showing selective binding of these antibodies to tumourcells.

The antibodies according to the invention recognize the peptidecorresponding to CD44 exon 6 having the amino acid sequence shown inFIG. 7, its allel variations and phosphorylation and glycosylationproducts and characteristic fragments thereof. Such antibodies arespecific to the peptide corresponding to CD44 exon 6 also in thepresence of other peptides which correspond to other CD44 exons. Fortherapeutic purposes this specificity is defined to the effect that theantibody according to the invention binds only to a little extent toproteins other than the protein encoded by exon 6. This unspecificbinding must be so little as to ensure that no considerable damage willbe caused to healthy cells when the antibodies according to theinvention are used for tumour therapy or in vivo diagnosis.

The antibodies can be used as whole antibodies, fragments thereof (e.g.Fv, (Fv)₂, Fab, Fab', F(ab)₂, chimeric, humanized or human antibodies aslong as they are binding the exon 6 protein in a suitable manner.Short-chain antibody fragments containing only the CDR regions or partsthereof conferring the specific binding to the exon 6 peptide are alsosuitable, especially if the antibody is a labelled one.

Here the antibodies can be used as a whole for therapy of malignantdiseases (Hale et al., Lancet 2 (1988) 1394-1399; Cobbold et al., Prog.Clin. Biol. Res. (1990) 333, 139-151). In another approach, the antibodyor part of it is conjugated or translationally fused to a toxin molecule(immunotoxin), thus effecting specific killing of tumour cells(Brinkmann et al. 1991, Proc. Natl. Acad. Sci. USA 88, 8616-8620; Pastanet al. (1991), Cancer Res. 51, 3781-3787; FitzGerald and Pastan (1989),J. Natl. Cancer Inst. 81, 1455-1461). In another preferred embodiment ofthe invention, bispecific antibodies are used for tumour therapy (Boninoet al. (1992), BFE 9, 719-723), which may be constructed by in vitroreassociation of polypeptide chains, by hybrid hybridoma generation orby construction of diabodies (Holliger et al. (1993), Proc. Natl. Acad.Sci. USA 90, 6444-6448; Holliger and Winter (1993), Current Opin.Biotechnol. 4, 446-449).

In addition, antibodies coupled to radioactive or fluorescent substancesare preferred for detection and treatment of tumours, includingcarcinomas of the respiratory, gastrointestinal and urogenital system aswell as ocular and skin cancers (Profio (1988), Proc. Soc. Photoopt.Instr. Eng. 907, 150-156; Jiang et al. (1991), J. Natl. Cancer Inst. 83,1218-1225).

For prevention of an immune response, it is preferred to use antibodieswhich resemble as closely as possible antibodies of human origin (Glassyand Dillman (1988), Mol. Biother. 1, 7-13). Such antibodies are, forexample, chimeric or humanized (CDR-grafted) antibodies. Such antibodiesusually are manufactured from a rodent monoclonal antibody (see e.g. forreview: Morrison (1992), Annu. Rev. Immunol. 10, 239-265; Winter andMilstein (1991), Nature 349, 293-299). In a specifically preferredembodiment of the invention, tumour specific human antibodies(Borrebaeck et al. (1988), Proc. Natl. Acad. Sci. USA 85, 3995-3999;Borrebaeck (1988), Immunol. Today 9, 355-359) are used for therapeuticpurposes. In addition, it is specifically preferred to prepare humanMabs via phage display libraries, as is described, for example, byGriffith et al., EMBO J. 12 (1993) 725-734.

It is specifically preferred to use, for therapeutic purposes,antibodies which impart effector functions (ADCC, CDC) (Bruggemann etal., J. Exp. Med. 166 (1987) 1357-1361). Particularly preferably, ahuman isotype IgG 1 antibody is used.

With regard to immunotoxins, it is preferred to couple the antibodyaccording to the invention to a toxin, such as, for example, Pseudomonasexotoxin, Diphtheria toxin or other toxins (FitzGerald and Pastan(1989)). It is also preferred to couple the antibodies tochemotherapeutics, such as, for instance doxorubicin, or toradioactively labelled substances which have a cytotoxic effect.

Conjugates of the antibodies according to the invention, in particularof human antibodies, for in vivo imaging, using, for instance,radioactive or fluorescent substances, are also preferred.

The therapeutic compounds of this invention may be administeredparenterally, such as intravascularly, intraperitoneally,subcutaneously, intramuscularily, using forms known in thepharmaceutical art. The active drug components of the present inventionare used in liquid, powdered or lyophilized form and may be combinedwith a suitable diluent or carrier, such as water, a saline, aqueousdextrose, aqueous buffer, and the like. Preservatives may also be added.

Regardless of the route of administration selected, the compounds of thepresent invention are formulated into pharmaceutically acceptable dosageforms by conventional methods known to those skilled in the art. Thecompounds may also be formulated using pharmacologically acceptable acidor base addition salts. Moreover, the compounds or their salt may beused in a suitable hydrated form.

Regardless of the route of administration selected, a non-toxic buttherapeutically effective quantity of one or more compounds of thisinvention is employed in any treatment. The dosage regimen for treatingis selected in accordance with a variety of factors including the type,age, weight, sex and medical condition of the patient, type of tumour,the route of administration and the particular compound employed in thetreatment. A physician of ordinary skill can readily determine andprescribe the effective amount of the drug required regarding knownantibody therapy approaches (Hale (1988), Cobbold (1990)). In soproceeding, the physician could employ relatively low doses at first,and subsequently, increased dose until a maximum response is obtained.

The chaotic over-expression of multiple spliced variants of the CD44gene in tumours, implies that a particular exon may or may not beover-expressed (or expressed at all) by a particular tissue sample. Animmunoassay using an antibody to the peptide expressed by any singleexon may therefore give misleading results. This invention thereforeincludes use, for the immunological diagnosis of neoplasia, of a mixtureof antibodies to two or more, and preferably to all nine, of the CD44exons.

In the examples which follow it was found that expression of the humanCD44 gene was consistently and distinctively increased in various solidtumours relative to normal tissues. Malignant (i.e. already metastatic)tumours differed from locally invasive and benign ones in the patternand magnitude of changes seen. The study was performed on samples from46 tumours of which 44 were locally invasive, or metastatic and 2 werebenign. Analysis of CD44 expression was performed by using PCR toamplify cDNA made by reverse transcription of RNA extracted from freshsurgical biopsy samples. By choosing oligonucleotide primers whichspecifically anneal to certain portions of the CD44 gene, it is possibleto amplify portions of the gene which, from these results, are ofdiagnostic and prognostic interest.

The strong association found here, between altered CD44 expression andneoplasia, need not imply that any of the individual exons of the geneare expressed only in neoplasia or in progression to metastaticmalignancy. Evidence accrued in many laboratories in recent years (seeKnudson 1985, Tarin 1992, Hayle et al 1992 for reviews) indicates thatthese pathological processes are probably the consequences of disturbedregulation of genes coding for normal cellular activities such as cellproliferation and migration. Therefore it seems unlikely that any gene,or portion of a gene, has the sole function of programming neoplasia ormetastasis.

The finding in the present study of transcripts from exon 10/11 innormal tissues, indicates that this exon is not exclusively concernedwith metastatic activity, even though there is marked increase in thenumber and signal intensity of bands hybridising with radiolabelledprobe E4 in the PCR products from tumours capable of metastasis. Othersupporting events are therefore believed to be required for CD44 exon10/11 expression to result in metastatic behaviour. Nevertheless, theobservation that transcripts from this exon were over-expressed insamples from metastatic tumours promises to be a very useful indicatorof prognosis.

It is not expected that further research will find that the natural(non-mutated) products of any individual exon will be uniquely presentin tumour cells and not in normal counterparts. Instead, it is likelythat an abnormal pattern of gene activity consisting of over-expressionand inappropriate combination of products of a gene, such as thatreported here for the CD44 locus, could play a part in malignancy. Thesechanges may themselves be required for malignant conversion, or be theconsequence of other genetic disturbances causing such a conversion.Even so, without resolving this issue, an observer using thesetechniques can obtain information relevant to assigning a sample toneoplastic or non-neoplastic categories.

EXAMPLES

Method

Fresh tissue samples, 0.5-1 cm diameter, were obtained from surgicalresection specimens removed at therapy of 34 patients with breasttumours and colon tumours. The samples were snap-frozen in liquidnitrogen within ten minutes of arrival in the pathological specimenreception area and kept in liquid nitrogen until use. Portions of lymphnode metastases and blood-borne metastases were also collected, ifpresent, in the tissue resected for diagnosis. Normal breast tissue,normal colon mucosa, normal lymph node adjacent to the tumour in thebreast and normal liver were also collected from the surgically resectedsamples and from other samples removed for non-neoplastic conditions.Normal peripheral blood leukocytes were obtained from 10 volunteers andbone marrow from 3 volunteers. The histological features of the tumoursand their clinical stages were as described in Table 1.

Total cellular RNA extraction from tissue samples was performedaccording to the method described by Chomizynski and Sacchi (1987).Extraction from fluid samples was by use of the Microfasttrack kitmarketed by Invitrogen. cDNA synthesis and subsequent amplification bythe polymerase chain reaction (PCR) was performed using the Superscript™preamplification system (BRL Life Technologies Inc., Middlesex, UK) withbuffers and reagents supplied in this kit. In brief, this involves aninitial step of first strand cDNA synthesis with reverse transcriptase,using sample RNA as the template and supplied nucleotide triphosphates.For subsequent PCR each sample was overlaid with oil and heated at 94°C. for 5 minutes to denature nucleic acid; 30 cycles of PCR were thenconducted with the following cycle parameters: 94° C. for 1 m, 55° C.for 1 m, 72° C. for 2 m. Negative controls in which there was notemplate cDNA in the reaction mix, were routinely run with each batch.The primers and probe sequences we devised, using information from thepublished sequence for human CD44 cDNA (Hofmann et al, 1991, Stamenkovicet al, 1991, Jackson et al, 1992) (FIG. 6) were as follows:

P1=5'GACACATATTGCTTCAATGCTTCAGC

P4=5'GATGCCAAGATGATCAGCCATTCTGGAAT

P1 is located with its origin 324bp upstream from the insertion site inthe standard CD44 molecule (between nucleotides 782 and 783 in thesequence published by Stamenkovic et al, 1989) and P4 is 158 bpdownstream of this site. These primers produce a PCR fragment of 482 bpif a sample expresses standard CD44 (so-called haemopoletic CD44), 878bpfor the epithelial form of CD44 and several other bands, if a samplecontains alternatively spliced transcripts. 10 μl of each PCR productwas electrophoresed in a 1.2% agarose gel and transferred to Hybond N⁺(Amersham UK, Little Chalfont, UK) nylon membranes for hybridisationwith oligonucleotide probe E4 (=5'TGAGATTGGGTTGAAGAAATC-3'), see FIG. 6.Blotting and autoradiography were performed to improve sensitivity ofdetection and resolution. The probe was radiolabelled with y³² P-ATP inthe presence of polynucleotide kinase. After prehybridisation,hybridisation was performed in 10% dextran, 6×NET, 5×Denhardt solution,0.5% NP40 and 100 μg/ml salmon sperm DNA at 42° C. overnight. The filterwas then washed twice in 2×SSC, 1×SSC and 0.5% SSC with 0.1% SDS at 42°C. sequentially for 15 minutes each. Filters were exposed to Kodak X-rayfilm for 2-16 hours. After this, the filters were boiled in 0.5% SDS forstripping the probe and rehybridised with another radiolabelled probe,namely P2 (=5'CCTGAAGAAGATTGTACATCAGTCACAGAC) we designed to anneal tothe standard portion of the CD44 (FIG. 6). The conditions used forhybridisation, washing and autoradiography were the same as above.

Calibration of the sensitivity of the method, for detection of smallnumbers of cells was performed as follows: total peripheral bloodleukocytes (PBL) were purified from 20 ml whole blood by lysis of packedred blood cells by addition of ammonium chloride buffer (1 ml packedcells to 50 mls lysis buffer) and subsequent centrifugation 15 minuteslater. The white cell pellet was divided into 4 tubes which were seededrespectively with 0 μl, 1 μl, 10 μl and 100 μl of a suspension of HT29colon carcinoma cells (5000 cells per ml). Total RNA was then extractedand each tube yielded approximately 20 μg. cDNA synthesis was performed,as described above on 4 μg aliquots of the RNA obtained from each tuberepresenting 0, 1, 10 and 100 tumour cells per aliquotted samplerespectively. The PCR was performed on these samples and on positive(tumour cells only) and negative (no DNA) controls using primers D1 andD5 which were designed by us to anneal specifically to exons 7 and 14 inFIG. 6. We know from previous studies that HT29 cells express bothexons, and others, in a pattern easily distinguishable from PBL andchose the oligonucleotide primers D1 and D5 because we wished toincrease sensitivity by shortening the segment to be amplified. It wasalso reasoned that use of these primers would circumvent the problem ofusing primers P1 and P4 for this specific purpose because the majorityof these would be soaked up by annealing to the standard portion of thegene. PCR cycle parameters, blotting, probing and washing conditionswere as described above. The oligonucleotide sequence used for probingwas ³² P labelled E4.

General Overview of Results

As the primers (P1 and P4) amplify across the splice product insertionsite it is clear that the intervening part of the standard molecule willbe amplified, in addition to any alternatively spliced variants whichcontain transcriptexfrom the additional exon domains. Hence the totalnumber of products which could conceivably be detected with a probe(e.g. P2) to the standard form considering all possible combinations ofthe sequences identified from this locus, is large. Using probe E4, 16of these combinations, namely those containing E4 transcripts from exon11, could potentially be visualised as bands of different molecularsizes resolved by electrophoresis. In practice the full range ofpossible combinations was not detected in these results, but several (upto 9) alternative splice variants were seen in neoplastic tissueshybridised with each probe. Normal tissues from the breast, colon andlymph nodes did express some E4-containing transcripts (FIGS. 1 and 3),in addition to the standard molecule (FIGS. 2 and 4), but peripheralblood leukocytes (FIG. 5) and liver (FIG. 4) detectably expressed onlythe latter with this combination of probes and primers. The details arepresented below:

Example 1

Breast Tissue Samples

The results obtained in the study of breast tissue samples areillustrated in FIGS. 1 and 2. Metastatic tumour deposits and theircorresponding primary tumours from all cases over-expressed severalalternatively spliced products containing transcripts from exon 11 (FIG.1a). At least 8 separate bands were frequently seen together with aconsistent doublet at 1500 bp and 1650 bp present in all tumours. Normalbreast tissue and normal lymph node produced two bands (1150 bp and 860bp) with this probe. The doublet mentioned above was not seen in anynormal sample.

The differences between the number, and size of the bands and theintensity of signal from the bound probe, between tissues in normal andmalignant categories, was obvious in all samples examined. Foroccasional samples it was necessary to expose the filter to the X-rayfilm for longer, to see the distinctive differences, but this findingwas confirmed in every case studied.

Samples from locally invasive tumours with no associated clinicalevidence of metastasis and from the two fibroadenomas alsoover-expressed splice products containing transcripts from exon 10/11relative to normal tissues, but the extent of this was easilydistinguished from the results obtained with malignant tumours and theirmetastases. Distinction from the patterns seen in normal tissues wasalso easy (FIG. 1b). However, a single sample gave a similar result tomalignant tumours (lane 14) (see below). The two fibroadenomas showedband patterns that were similar to those from non-metastatic carcinomasand the sample from a case of cystic disease of the breast resembled thepattern for normal non-neoplastic breast tissue. This is the firstinstance of definitive diagnosis by this method. The piece of tissue wasprovided by the duty pathologist as being from a benign tumour, namely afibroadenoma, on macroscopic appearance at initial inspection with thenaked eye. It was then characterised as definitely non-neoplastic afterPCR amplification of its cDNA, and subsequent microscopical examinationof the tissue confirmed this.

To confirm that the differences seen with probe E4 are valid and nottechnical artifacts, the results obtained when the same filter washybridised with probe P2 are shown in FIG. 2. This shows that i) alltissues examined expressed the standard form of the gene, ii) other exonsplice products, not containing transcripts from exon 10/11, werepresent in tumours and metastases and iii) that the differencesdescribed above are not due to unequal loading of tracks in the variouspanels and lanes on this composite filter, but resulted from alternativesplicing. All conditions in this experiment were the same as those inhybridisation with E4, except the exposure time of the filter to X-rayfilm (10 hours exposure for FIG. 1, versus 1.5 hours for FIG. 3).

Example 2

Colon Samples

The findings in colon carcinoma were identical to those in breastcarcinoma. Thus, in all cases the colon carcinoma tissues showedincreased number of more intensely labelled, larger molecular weightbands with probe E4 (FIG. 3) than normal colonic mucosa and other normaltissues. As with breast carcinomas, hybridisation with probe P2 showedno differences in the degree of expression of the standard form of themolecule (FIG. 4).

Example 3

Calibration of the Sensitivity of the Method

Examination of autoradiograms of PCR products of peripheral bloodleukocytes seeded with known numbers of HT29 colon carcinoma cellsshowed the presence of additional bands characteristic of tumour cells,down to a level of 10 tumour cells in a sample of 10⁷ leukocytes. Byfine-tuning the conditions of the assay it is considered possible todetect a single tumour cell in 10 ml of blood.

In the series described above, all samples of neoplastic tissue showedover-expression of alternatively spliced products of the CD44 gene andnone of the samples from non-neoplastic tissue did so. Therefore, therewas complete correspondence between normal or neoplastic origin of asample and pattern of CD44 expression. In one instance, a tumour removedfrom a patient (patient B16, lane 14 in FIG. 1A) with no currentclinical evidence of metastasis, was found to have a pattern ofexpression indicating metastatic capability. At present it is notpossible to know whether this is a false positive result, or a sign ofimminent metastasis. This patient is currently under observation in thefollow-up clinic.

Example 4

We have designed and synthesised oligonucleotide primers according toour current findings, as follows:

Primer P1=5'-GACACATATTGCTTCAATGCTTCAGC (458-484)

Primer P2=5'-CCTGAAGAAGATTGTACATCAGTCACAGAC (488-518)

Primer P3=5'-TGGATCACCGACAGCACAGAC (746-767)

Primer P4=5'-GATGCCAAGATGATCAGCCATTCTGGAAT (912-941) for standard part(Stamenkovic 1989)

Primer E1=5'-TTGATGAGCACTAGTGCTACAGCA

Primer E2=5'-CATTTGTGTTGTTGTGTGAAGATG

Primer E3=5'-AGCCCAGAGGACAGTTCCTGG (534-554)

Primer E4=5'-TGAGATTGGGTTGAAGAAATC (558-578)

Primer E5=5'-TCCTGCTTGATGACCTCGTCCCAT (585-608)

D1: 5' GAC AGA CAC CTC AGT TTT TCT GGA (63-86)

D5: 5' TTC CTT CGT GTG TGG GTA ATG AGA (888-911)

for the exons (Hofmann 1991). E1 and E2 are on exon 6.

Fresh tissue samples 0.5-1 cm in diameter were obtained from surgicalresection specimens or at autopsy. All samples used in this work wereobtained from the residue of tissue remaining after diagnostic sampleshad been taken, and which would otherwise have been discarded. Thesamples were snap-frozen in liquid nitrogen within ten minutes ofarrival at the pathological specimen reception area and kept frozen innitrogen until use. cDNA was synthesised with viral reversetranscriptase using 5 μg of total cellular RNA as template, followed byPCR with Primer P1 and Primer P4. PCR amplification, electrophoresis andhybridisation were performed under standard conditions.

When the PCR products were hybridised with radiolabelled E2 or E4, allsamples from carcinomas over-expressed several splice variants, but thepattern of bands seen with each probe was different. Hence, theoligonucleotide probe for Exon 6 products is very effective indistinguishing neoplastic from non-neoplastic samples, but notsignificantly more sensitive than E4, at least on samples from solidtissues, but is possibly useful for detecting organ of origin of adisseminating metastatic cell or an established metastasis.Subsequently, the same filters were stripped and hybridised with P2probe to show that all samples, including normal tissues, produced thestandard portion of CD44. This confirmed that the differences observedbetween the results obtained with normal and tumour samples, probed withE2 and E4, were not due to unequal loading of PCR products. Thecumulative results are summarised in Table 3 which indicates that thesechanges are seen in a wide range of common cancers.

                  TABLE 3    ______________________________________                  No. of Patients/                             No. Showing Increased    Type of Tissue                  Volunteers Splice Variants    ______________________________________    Neoplastic    47         46    Breast Cancer 21         21    Colon Cancer  13         13    Bladder Cancer                  6          6    Stomach Cancer                  1          1    Thyroid Cancer                  1          1    Fibroadenoma  2          2    Prostate Cancer                  3          2    Non-Neoplastic                  39         0    Normal Breast 9          0    Cystic Disease of Breast                  1          0    Normal Colon  9          0    Crohn's Disease                  1          0    Ulcerative Colitis                  1          0    Appendicitis  1          0    Normal Bladder                  4          0    PBL           10         0    Bone Marrow   3          0    ______________________________________

We have also examined some malignant tumours of bone muscle and observeda similar pattern, of marked over-expression of multiple splicedvariants, in the osteosarcoma.

Example 5

Cancer Diagnosis by PCR Assay of Clinically-Harvested Urine Samples

Approximately 50 ml naturally-voided urine were obtained from eachperson and transported to the laboratory as speedily as possible.Specimens from 90 patients were examined: 44 from patients withbiopsy-proven bladder cancer, 46 from patients with non-neoplasticinflammation of the bladder (cystitis) and from normal volunteers. Oneml of each urine sample was removed after thorough mixing and submittedfor cytological examination. Another 1 ml of urine was checked byFluorescein diacetate-ethidium bromide staining to assess the viabilityof cells in the sample. The remainder of the urine was centrifuged at2000 rpm for 10 minutes and the cell pellet was kept at -70° C. untiluse. mRNA extraction was performed with oligo dT cellulose tablets(invitrogen). cDNA was synthesised with AMV reverse transcriptase(Invitrogen). The completed cDNA solution was divided equally into twotubes, one being for PCR with E1 and E5, to amplify the particular cDNAtranscript, which we have found to be of diagnostic value and the otherfor PCR with P1 and P4 to amplify the standard form of CD44, with orwithout all splice variants, as an internal control.

Thirty-five cycles PCR were then carried out. The cycle conditions were:95° C. 1 minutes, 55° C. 1 minute, 72° C. 2 minutes. A hot startprocedure was adopted for all samples. Results are shown in FIG. 8.

Equal volumes of PCR products were loaded in each lane of a 1.2% agarosegel and stained with ethidium bromide. If the cells in the urine were tobe expressing all the Exons from Exon 6 to Exon 14, it was predictedthat with the current PCR protocol, using primers E1 and E4, shouldproduce a 735 bp band. There is no band in tracks containing cDNA fromnormal urine or that of patients with non-neoplastic cystitis (lanes1-8) but a clear 735 band is seen in all urine samples from patientswith bladder cancer (lanes 9-16) when PCR was performed with primer E1and E5 (upper panel).

A 482 bp band representing the standard form of CD44 was obtained almostequally in all cases when PCR was performed with P1 and P4 (lowerpanels). This indicates that the diagnostically significant differencesbetween urine from patients with bladder cancer and that from controlswere not caused by unequal loading of the tracks but by alternativesplicing of the CD44 gene. Lanes 1-4: normal urine. Lanes 5-8: cystitisurine. Lanes 9-16: from patients 1-8 with bladder cancer.

In the overall results this 735 bp band was completely absent in 7 of 7normal and 9 of 9 cystitis-affected urine specimens; that is 0% falsepositive. Also 14 of 19 (74%) urine samples from patients with bladdercancer showed a positive result (i.e. 26% false negatives). In the falsenegative samples there was a shortage of viable cancer cells asindicated by fluorescein-d acetate ethidium bromide staining.

Example 6

Stools from 12 patients were assayed by the techniques described herein.Of the samples from 9 patients with colorectal carcinoma, 5 gavepositive results. Of the samples from 3 normal patients, all 3 gavenegative results. These figures, obtained from samples full of bacteriawhich were not subjected to any pretreatment, encourage the belief thata viable diagnostic assay could be developed without difficulty.

In the inventors' further experience of detecting tumour cells with thismethod, the following observations would be useful to othersinvestigating its diagnostic potential. The major considerations to beaware of are that the reliability and reproducibility of the resultsdepend critically on the quality of the mRNA obtained from the sampleand upon the care with which the techniques are performed. The mainrequirement is to eliminate false negative results by ensuring that highquality mRNA is routinely obtained and by using internal standards inevery reaction to monitor the PCR amplification step. False positives,providing they are not too frequent, are not a serious problem, becausethey can be recognised by replicate assays on the same or furthersamples and by reference to other clinical data.

The inventors have explored the procedures needed to ensure the routineRT-PCR detection of abnormal CD44 gene activity in small clinicalsamples containing tumour cells. If a tissue sample is divided intoaliquots, half of which are frozen in liquid nitrogen immediately andthe remainder of which are left at ambient temperature, one can show howthe ability to detect CD44 splice variants declines with time and withmode of specimen handling. Fresh samples submitted to mRNA extractionwithin half an hour of excision give the most reliable results and thereis a gradual decline in quality over the next few hours if the freshtissue is left at ambient temperature. If the sample is first snapfrozen, the results obtained when RNA is extracted immediately afterthawing are satisfactory, but decline very rapidly, beginning within 15minutes, the larger variant transcripts being lost first and ultimatelyeven the standard form. It is also found that if snap-frozen cell andtissue samples are stored at -70° C. the results decline after 4 weeks,even if the mRNA is extracted immediately after thawing. It would seemtherefore that degradation of RNA by ribonucleases released from cellsruptured during freezing continues, even at this temperature, althoughat slower rates. Further, as one would expect, if the sample taken forRNA extraction is from an area of necrosis or of fibrosis, one does notobtain the typical results seen with viable tumour tissue. Hence, carein sample selection and in specimen processing are both needed forgenerating reliable data.

Arising out of this, we prefer that a fresh sample should be held fornot more than 24 hrs before being either frozen or treated to extractmRNA; and that a thawed sample should be held for not more than 2 hrsbefore being treated to extract mRNA.

The diagnosis method described herein can be performed in a single day,possibly in a few hours, and is capable of being automated. Use of themethod has been demonstrated, on various tissue samples to detect awhole variety of cancers, and also on blood and urine samples. Wetherefore offer it as a convenient practical method for cancer screeningand diagnosis. In principle it could also have wide generalapplicability to cancer detection and prevention programmes andtherefore have epidemiologic and public health value. Proper applicationof its sensitivity, specificity and simplicity should add not only toinitial cancer diagnosis but to evaluation of extent of disease in thebody, to judgment of the efficacy of treatment and to early detection oftumour recurrences.

FIGURE LEGENDS

Notation: N=normal, T=primary tumour, M=metastasis.

FIG. 1

Autoradiogram of PCR products from breast tissue samples probed with E4(10 hours exposure of X-ray film to sample filter). Panel A: malignantprimary breast carcinomas with their metastases. Tracks 1, 2 and 3:patient B1; tracks 4, 5 and 6: patient B2; tracks 7, 8 and 9: patientB3; tracks 10 and 11: patient B4; tracks 12 and 13: patient B5. It canbe seen that compared to normal breast tissue, primary breast carcinomasand their metastatic deposits overexpress several splice-variants. Notethe doublet (arrows) at 1500 bp and 1650 bp best seen in track 5. Thisis present in all tumours and metastases but is fogged in the othertracks by this time of exposure. It is not detectable in any normalsamples even at much longer exposure times (23 hours). Panel B: Breastcarcinomas with no clinical evidence of metastasis. Tracks 14-20 arefrom patients B15-B21. The tumours all overexpress several variants, butshow less bands and the signal intensity is less, except track 16(patient B17)--see text. The 1500/1650 bp doublet (arrow) is easilyrecognisable in tracks 15, 16 and 18 at this length of exposure andbecame detectable in all other tumour-containing tracks on longerexposure. The illustration, however, shows only the shorter exposure, toavoid fogging the tracks which have stronger signals. Panel C:Fibroadenomas (FA) and fibrocystic disease of the breast (Cyst). Tracks21 and 22, containing the benign tumour samples (samples B22 and 23),express more than the non-neoplastic sample (fibrocystic disease) intrack 23 (sample B24).

FIG. 2

Autoradiogram of PCR products from breast tissue samples probed withprobe P2 (1.5 hours exposure of X-ray film to sample filter). Thisresult was obtained by reprobing the same filter as that used in FIG. 1,after stripping off the previous probe. Here it can be seen that i) thedifferences observed in FIG. 1 are not due to unequal loading of tracks,ii) that the expression of the standard form of the molecule isquantitatively greater than any of the variants, iii) the standard formis expressed in all tissues examined and iv) further variants which donot contain exon 3 transcripts, are also present and over-expressed intumours. The 1500/1650 bp doublet can be recognised in the tumours inpanel A but needed longer exposure to be detectable in panels B and C.

FIG. 3

Autoradiogram of PCR products from colon tissue samples probed with E4(10 hours exposure of photographic film to sample filter). Tracks 1, 2and 3: patient C1; tracks 4, 5 and 6: patient C2; tracks 7, 8 and 9:patient C3; tracks 10 and 11: patient C4; tracks 12 and 13: patient C5;track 14: normal liver sample. The picture shows the same features asdescribed in the legend to FIG. 1 and that the findings apply tocarcinomas of the colon. The 1500/1650 bp doublet (arrow) is easilyrecognisable in several tumour tracks (2 and 8-12) and the faint signalin the corresponding position in tracks 3, 5, 6 and 13 became strongeron longer exposure. However none appeared in this vicinity in tracks 1,4, 7 or 14 (normal tissue).

FIG. 4

Autoradiogram of PCR products from colon tissue samples probed with P2(1.5 hours exposure of photographic film to sample filter). Thisconfirms equal loading of the tracks and that other points, illustratedin FIG. 2, apply to colon carcinomas. Note that normal liver expressesthe standard form of CD44.

FIG. 5

Autoradiogram of PCR products of normal peripheral blood leukocytes, PBL(from 3 different persons) and other normal tissues probed with E4(panel A; 8 hours exposure to photographic film) and P2 (panel B; 5hours exposure to photographic film). Track 6 contains PCR products froma breast cancer (patient B1) as a positive control. With thiscombination of primers and probes, leukocytes can be seen to express thestandard form of the CD44 molecule, but no detectable splice variants.The samples in tracks 4 and 5 were from individuals with no clinicalevidence of neoplasia, as follows: track 4, breast tissue obtained atautopsy from the body of a woman who died of bacterial endocarditis;track 5, colon resected for volvulus.

                                      TABLE 1    __________________________________________________________________________    PATIENT         AGE            DISEASE                   TUMOUR SIZE                           METASTASIS                                    HISTOLOGY (GRADE)                                               CLINICAL STAGE    __________________________________________________________________________    B1   56 Breast ca                   2.5 cm  Lymph node    B2   53 Breast ca                   3   cm  Lymph node    B3   65 Breast ca                   3   cm  Lymph node    B4   54 Breast ca                   5   cm  Lymph node (10/10)                                    IDC (mucinous)  1!    B5   59 Breast ca                   5.5 cm  Lymph node    B6   59 Breast ca                   3   cm  Lymph node    B7   61 Breast ca                   4   cm  Lymph node (17/17)                                    ILC/IDC    3    B8   38 Breast ca                   3.5 cm  Lymph node (1/5)                                    ILC        2    B9   65 Breast ca                   1.8 cm  Lymph node (5/6)                                    ILC        2    B10  61 Breast ca      Lymph node (10/13)                                    IDC  1!    2    B11  80 Breast ca                   11  cm  Lymph node                                    3    B12  65 Breast ca                   2.3 cm  Lymph node                                    ? 1    B13  68 Breast ca                   2.8 cm  Lymph node (4/12)                                    IDC  3!    2    B14  47 Breast ca                   7   cm  Lymph node          2    B15  38 Breast ca      None (0/7)                                    IDC        1    B16  62 Breast ca                   3   cm  None (0/4)                                    IDC  3!    1    B17  62 Breast ca                   3   cm  None (0/16)                                    IDC  2!    1    B18  63 Breast ca                   3   cm  None (0/16)                                    1    B19  61 Breast ca                   3   cm  None     1    B20  42 Breast ca                   4   cm  None     IDC        1    B21  65 Breast ca      Lymph node                                    IDC/ILC    B22  54 Breast ca                   6   cm  None (0/4)                                    IDC        1    B23  49 Fibroadenoma                   4   cm  --       --         --    B24  47 Fibroadenoma                   3   cm  --       --         --    B25  29 Cystic disease                   --      --       --         --    C1   72 Colon ca                   5.0 cm  Lymph node                                    Well diff. adeno                                               3  C!    C2      Colon ca       Lymph node    C3   65 Colon ca                   6.5 cm  Liver    Mod diff. adeno                                               4  D!    C4   56 Colon ca                   7.8 cm  Lymph node                                    Mod diff. adeno                                               4  D!    C5      Colon ca       Lymph node    C6   57 Colon ca                   5   cm  Lymph node                                    Mod diff. adeno                                               3  C!    C7      Colon ca       None    C8   75 Colon ca                   6.5 cm  Lymph node                                    Mod diff. adeno                                               3  C!    C9   72 Colon ca                   5.5 cm  Lymph node                                    Mod diff. adeno                                               3  C!    C10  76 Colon ca                   4.5 cm  None     Well diff. adeno                                               1  B!    T1      Thyroid ca    __________________________________________________________________________     Key:     IDC: infiltrating ductal carcinoma     ILC: infiltrating lobular carcinoma     Well diff. adeno: Well differentiated adenocarcinoma     Mod diff. adeno: Moderately differentiated adenocarcinoma     Letters in square brackets in Clinical Stage column refer to Dukes stagin     scheme for colon carcinoma

REFERENCES

1. Stamenkovic, Amiot M, Pesando J. M, Seed B. A lymphocyte moleculeimplicated in lymph node homing is a member of the cartilage linkprotein family. Cell 1989; 56: 1057-062.

2. Birch M, Mitchell S, Hart I. R. Isolation and characterisation ofhuman melanoma cell variants expressing high and low levels of CD44.Cancer Res. 1991; 51: 6660-6667.

3. Gunthert U, Hofmann M, Rudy W, Reber S, Zoller M, HauBmann, Matzku S,Wenzel A, Ponta H, Herrlich P. A new variant of glycoprotein CD44confers metastatic potential to rat carcinoma cells. Cell 1991; 65:13-24.

4. Sy M S, Guo Y-J, Stamenkovic I. Distinct effects of two CD44 isoformson tumor growth in vivo. J. Exp. Med 1991; 174: 859-866.

5. Hofmann M, Rudy W, Zoller M, Tolg C, Ponta H, Herrlich P, Gunthert U.CD44 splice variants confer metastatic behaviour in rats: Homologoussequences are expressed in human tumor cell lines. Cancer Res. 1991; 51:5292-5297.

6. Stamenkovic I, Aruffo A, Amiot M, Seed B. The hematopoletic andepithelial forms of CD44 are distinct polypeptides with differentadhesion potentials for hyaluronate-bearing cells. EMBO J. 1991; 10:343-348.

7. Jackson D. G, Buckley J, Bell J. I. Multiple variants of the humanlymphocyte homing receptor CD44 generated by insertions at a single sitein the extracellular domain. J. Biol. Chem. 1992; 267: 4732-4739.

8. Chomzynski P, Sacchi N. Single-step method of RNA isolation by acidguanidinium thiocyantat-phenol-chloroform extraction. Anal Biochem.1987; 162: 156.

9. Knudson A. G. Hereditary cancer, oncogenes and antioncogenes. CancerRes. 1985; 45: 1437-43.

10. Tarin D. Tumour metastasis. In: Oxford Textbook of Pathology 1992;(eds: J O'DMcGee, N. A. Wright, P. G. Isaacson). Oxford UniversityPress, Oxford. pp607-633.

11. Hayle A. J, Darling D. L, Taylor A. R, Tarin D. Transfection ofmetastatic capability with total genomic DNA from metastatic tumour celllines. Differentiation, 1993, in press.

12. Screaton G. R., Bell M. V., Jackson D. G., Cornelis F. B., Gerth U.,and Bell J. I., Genomic Structure of DNA encoding the lymphocyte homingreceptor CD44 reveals at least 12 alternatively spliced exons, Proc.Natl. Acad. Sci. USA, Vol 889, p 12160-4, December 1992, Immunology.

Example 7

I. Peptide Synthesis

5 peptides corresponding to amino acids 1-13, 9-23, 19-33, 29-43 and1-43 of the peptide sequence corresponding to CD44 exon 6 as shown inFIG. 7 were synthesized by 9-fluoroenylmethyloxycarbonyl (Fmoc)chemistry solid phase peptid synthesis (Atherton and Sheppard, 1989) onan Applied Biosystems, Inc., Model 431A Peptid Synthesizer using theproprietor's standard scale (0.25 mmol) Fmoc chemistry option. For thispurpose, 403 mg 4-(2', 4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyresin (Rink, 1987) with a substitution of 0.62 mmol/g resin are used.The amide resin is deprotected (Fmoc cleavage) by treatment with 20%piperidine in N,N-dimethyl formamide (DMF) before the first couplingcycle. For peptide synthesis, a 4-molar excess of the followingFmoc-amino acid derivatives and other carboxylic acids is used:

N-Fmoc-L-alanine

N-α-Fmoc-N^(G) -(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginine

N-α-Fmoc-N-β-(trityl)-L-asparagine

N-α-Fmoc-L-aspartic acid-β-t-butyl ester

N-Fmoc-S-trityl-L-cystein

N-α-Fmoc-N-gamma-(trityl)-L-glutamine

N-α-Fmoc-L-glutamic acid-gamma-t-butyl ester

N-α-Fmoc-N-im-trityl-L-histidine

N-Fmoc-L-leucine

N-α-butyloxycarbonyl-N-ε-Fmoc-L-lysine

N-α-Fmoc-N-ε-butyloxycarbonyl-L-lysine

N-Fmoc-L-norleucine

N-Fmoc-L-phenylalanine

N-Fmoc-L-proline

N-Fmoc-O-t-butyl-L-serine

N-Fmoc-O-t-butyl-L-threonine

N-α-FMoc-N-ε-butylocxycarbonyl-L-tryptophan

N-Fmoc-gamma-aminobutyric acid

N-Fmoc-ε-aminocaproic acid

(+)-Biotin

Prior to coupling, the amino acid derivatives are dissolved in DMF andactivated through the addition of 1 equivalent N-hydroxybenzotriazole(HOBt) in N-methylpyrrolidinone (NMP) and 1 equivalentN,N'-dicyclocarbodiimide (DCC) in NMP. The 20-minute couplings of theHOBt-ester amino acid are carried out in DMF. Following coupling,deprotection of the N-termini (Fmoc cleavage) is achieved by a 3-minuteand then a 10-minute treatment with 20% piperidine in DMF. The peptidechain is extended through repetition of theactivation/coupling/deprotection cycles. Peptides utilized later forimmunogen synthesis are outfitted with an N-terminal aminocaproic acidspacer and cystein, through which the peptide is tethered to the carrierprotein. For peptides used as screening reagents, a different N-terminusis synthesized and contains three gamma-aminobutyric acid moieties,lysine, and biotin (attached to the ε-amino group of lysine). Followingsynthesis, the peptide is removed from the resin support bytrifluoroacetic acid (TFA) cleavage. The peptide-bearing resin isreacted for 1 hour at room temperature (RT) with a cleavage cocktailcontaining 20 mL trifluoroacetic acid, 1 mL H₂ O, 1 mL thioanisole, 0.5mL ethanedithiol and 1.5 g phenol. Removal of the acid-labile side-chainprotecting groups, performed under Argon, is complete after anadditional 2.4 h reaction time at RT in the aforementioned cocktailsolution. After a brief cooling period, the deprotected peptide isprecipitated through the addition of diisopropylether. The precipitateis filtered, washed with dilsopropylether, dissolved in 50% acetic acid,frozen and lyophilized. Peptide purity is determined by reverse-phaseHPLC (column--Vydac 218TP54, C₁₈, 300 Å, 5 μm, 4.6×250 mm; mobilephase--A: 0.1% TFA in H₂ O, B: 0.1% TFA in H₂ O/acetonitrile (35/65,v/v); gradient--0-100% B in 90 min; flow rate--1 mL/min; detection--226nm). Those peptides being less than 60% pure are purified byreverse-phase HPLC (column--Waters DeltaPak C₁₈, 100 Å, 15 μm, 50×300mm; mobile phase--A: 0.1% TFA in H₂ O, B: 0.1% TFA in H₂ O/acetonitrile35/65, v/v, gradient--0.50% B in 130 min; flow rate--15 mL/min;detection--226 nm). Peptide identity is verified by plasma desorptionmass spectrometry. Characteristic HPLC retention times and mass spectraldata for the peptides used for immunogen synthesis are listed in Table2.

                                      TABLE 2    __________________________________________________________________________    HPLC and MS Characteristics of Activated    Hapten Peptides                                 Exp.    Retention                             Theoret.                                 mass                                     Exp.                                         time    Peptid Name  Sequence.sup.1                             mass                                 (MH+)                                     Theoret                                         (min).sup.2    __________________________________________________________________________    AH,CD44(AT1-13NH.sub.2,1-ZC)                 H-CZATTLJSTSATAT                             1651.76                                 1653.3                                     +1.54                                         36.01                 ETA-NH.sub.2    AH,CD44(9-23NH.sub.2,9-ZC)                 H-CZATETATKRQETW                             2155.31                                 2155.8                                     +0.49                                         45.29                 DWF-NH.sub.2    AH,CD44(19-33NH.sub.2,19-ZC)                 H-CZTWDWFSWLFLPS                             2214.47                                 2215.7                                     +1.23                                         61.73                 ESK-NH.sub.2    AH,CD44(29-43NH.sub.2,29-ZC)                 H-CZPSESKNHLHTTT                             1950.15                                 1950.5                                     +0.35                                         28.92                 QJA-NH.sub.2    AH,CD44(1-43NH.sub.2,1-ZC)                 H-CZTLJSTSATATETA                             5150.5                                 5149                                     -1.50                                         57.64                 KRQETWDWFSWLFLP                 SESKNHLHTTTOJA-NH.sub.2    __________________________________________________________________________     .sup.1 J = norleucine, 2, = aminocaproic acid, other abbreviations from     standardized oneletter code.     .sup.2 Retention times obtained using aforementioned HPLC conditions.

II. Activation of Carrier Protein

For immunogen synthesis, a carrier protein, either Keyhole LimpetHemocyanin (KLH) or Bovine Serum Albumin (BSA), is modified through theε-amine of lysines with the heterobifunctional cross-linking reagent,N-succinimidyl 3-maleimidopropionate (MPS). This imparts the carrierprotein with "handles" onto which the sulfhydryl peptides are laterconjugated. For the case of KLH, a 10 μM KLH solution is prepared with0.1M NaHCO₃, pH 8.35. The pH of the suspension is adjusted to 8.3 andbriefly centrifuged. After determining the protein concentration by thebicinchoninic acid (BCA) protein assay (Smith, et al., 1985), 3000equivalents of a 0.3M MPS solution in dimethylsulfoxide are addeddropwise to the stirred KLH solution and allowed to react at RT for 1hour. The solution pH is adjusted to 7.0 with 0.1M HCl, and activatedcarrier protein is separated from excess MPS by size-exclusionchromatography (column--AcA 202, IBF Biotechnics, 5×12 cm, RT;buffer--0.1M KH₂ PO₄ /K₂ HPO₄ pH 7.0, 0.1M NaCl; flow rate--6 mL/min,detection--226 nm). Protein concentration is again determined by the BCAProtein assay and the degree of maleimido-propionamide (MP) substitutionof the activated KLH (KLH-MP) is determined with the Ellman's reagent,DTNB (Ellman, 1959). For BSA, a 190 μM BSA solution is prepared in 0.1MKH₂ PO₄ /K₂ HPO₄ pH 7.0, to which is added dropwise 100 equivalents MPS(40 mM in 1,4-dioxane). After stirring the reaction mixture for 2 hoursat RT, it is loaded onto a size-exclusion column. The activated BSA(BSA-MP) is purified and analysed analogous to KLH-MP). Substitutionvalues of 20-35:1 and 200-600:1 are routinely achieved for the activatedcarrier proteins, BSA-MP and KLH-MP, respectively.

III. Conjugation of Peptide with Activated Carrier Protein.

Through formation of a thioether bond, thiol-containing peptides areconjugated with the MP-activated carrier protein. In the case of BSA-MP,a 74 μM BSA-Mp solution in 0.1M KH₂ PO₄ /K₂ HPO₄ pH 7.0 is reacted with1 equivalent (with respect to MP) of a 4 mM peptid solution in the samephosphate buffer. The solution is stirred slowly and allowed to react atRT overnight. After centrifugation, the soluble BSA-MP-peptide conjugateis separated from unbound peptide via size-exclusion chromatography(same chromatography conditions as given in section II). Analyses of theprotein conjugate include protein concentration determination via BCA,as well as ascertaining the remaining number of unreacted MP-groups withEllman's reagent. KLH-MP-peptide conjugates are synthesized similarlywith the exception of activated carrier protein and peptideconcentrations, which are 3 μM and 18 μM, respectively.

REFERENCE

Atherton, E. and Sheppard, R. C. (1989) Solid Phase Peptide Synthesis: APractical Approach, Oxford, U. P., Oxford.

Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77.

Rink, H. (1987) Solid-Phase Synthesis of Protected Peptide FragmentsUsing a Trialkoxydiphenylmethylester Resin. Tetrahedron Letters 28,3787-3790.

Smith P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F.H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. andKlenk, D. C. (1985) Measurement of Protein Using Bicinchoninic AcidAnal. Biochem. 150, 76-85.

ABBREVIATIONS

BCA--bicinchoninic acid

BSA--bovine serum albumin

BSA-MP--bovine serum albumbin activated with N-succinimidyl3-maleimidopropionate

DCC--N,N'-dicyclocarbodiimide

DMF--- N,N-dimethylformamide

DTNB--dithio-bis-(2-nitrobenzoic acid), Ellman's reagent

Fmoc--9-fluorenylmethyloxycarbonyl

HOBt--N-hydroxybenzotriazole

KLH--Keyhole Limpet hemocyanin

KLH-MP--Keyhole Limpet hemocyanin activated with N-succinimidyl3-maleimidopropionate

MP--maleimidopropionamide

MPS--N-succinimidyl 3-maleimidopropionate

NMP--N-methylpyrrolidinone

RT--room temperature

TFA--trifluoroacetic acid

Example 8

Manufacture of the recombinant HIV2 (gp32)-CD44 exon 6 antigen/immunogen

Exon 6 of the CD44 gene codes for a peptide of 43 amino acids as shownin FIG. 7.

Peptides and small proteins of less than 100 amino acids as a rulecannot be made recombinantly by cytoplasmic expression in amicroorganism. For this reason, a fusion gene comprising a gene which isreadily expressible in E. coli (part of the envelope protein gp32 of theHIV2 retrovirus) and CD44 exon 6-DNA was constructed.

To increase the CD44 exon 6 epitopes in the fusion protein, the CD44exon 6 antigen was duplicated at the DNA level using a suitable linker(codes for the 3 C-terminal amino acids of the exon 5 of CD44).

Six histidine residues (codons) were inserted in the N-terminal regionof the HIV2 (gp32)-CD44 exon 6 fusion protein at the DNA level for thepurpose of simpler antigen/immunogen isolation by means of metal chelateaffinity chromatography.

Recombinant DNA Technique

Standard methods were used to manipulate the DNA such as those describedby Sambrook, J. et al. (1989) In: Molecular cloning: A laboratorymanual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Construction of HIV2 (gp32)-partial gene (plasmid pUC18₋₋ HIV2-gp32)

The coding section of the amino acid 48-162 of the HIV2-gp32 gene wassynthesized by overlapping chemical gene synthesis and subsequentlysubcloned in the plasmid pUC18. Production and description of theplasmid pUC18₋₋ HIV2-gp32 are described in the European PatentApplication 0 440 207.

Construction of the E. coli expression vector pDS56-6HIS-HIV2-gp32

In the following plasmid construction, a fusion gene was constructedwhich codes for the N-terminal of the amino acid sequenceMRGSHHHHHHTDPEF (poly-His tail) and the selected HIV2-g 32 antigen.

For this purpose, the vector pQE-10 was digested with restrictionendonucleases BamHI and HindIII, and the approx. 3.4-kbp-longBamHI/HindIII-pQE-10 vector fragment isolated by agarose gelelectrophoresis. The pQE-10 vector synonym: pDS56/RBSII, 6×His(-1)! issourced from Diagen, Germany, and is described in Stuber, D. et al.(1990) Immunol. Methods IV: 121-152. In a second preparation, theplasmid pUC18₋₋ HIV2-gp32 was digested by the restriction endonucleasesBamHI and HindIII, and the ca. 400-bp-long BamHI/HindIII-HIV2-gp32fragment isolated and ligated into the approx. 3.4-bp-longBamHI/HindIII-pQE-10 vector fragment. The desired plasmid was identifiedby restriction mapping and designated pDS56-6HIS-HIV2-gp32.

Construction of E. coli expression vector pDS56-HIV2-CD44 exon 6

In the following plasmid construction, a fusion gene was constructedwhich codes N-terminal for the amino acid sequence MRGSHHHHHHTDPEF(poly-His tail), the selected HIV2-gp 32 antigen, and 2 copies of theCD44 exon 6 antigen.

Two copies of the CD44 exon 6 gene were produced by polymerase chainreaction (PCR) Mullis, K. B. and Faloona, F. A. (1987) Methods Enzymol.155: 335-350!.

In a first PCR reaction, the CD44 exon 5-6 DNA sequence from base pairposition 397-538 was amplified (see FIG. 9: DNA sequence ofHIV2(gp32)-CD44 exon 6 fusion gene) and provided with suitable singularrestriction endonuclease cleavage sites (BamHI and HaeIII). SubclonedCD44 cDNA (Exon 5-11) and the following primer pair were used foramplification: ##STR1##

The approx. 160-bp-long PCR product was digested with BamHI and HaeIIIand the approx. 150-bp-long BamHI/HaeIII-CD44 exon 6 fragment isolatedby agarose gel electrophoresis.

In a second PCR reaction, the CD44 exon 5-6 DNA sequence of base pairposition 539-672 (see FIG. 9: DNA sequence of HIV2(gp32)-CD44 exon 6fusion gene) was amplified and provided with suitable singularrestriction endonuclease cleavage sites (HaeIII and HindIII) usingsubcloned CD44 cDNA exon 5-11 as template DNA and the following primerpair: ##STR2##

The approx. 150-bp-long PCR product was digested by HaeIII and HindIIIand the approx. 140-bp-long BamHI/HaeIII-CD44 (Exon 6) fragment isolatedby agarose gel electrophoresis.

Then the BamHI/HaeII-CD44 exon 6 fragment from the first PCR reactionand the HaeIII/HindIII-CD44 exon 6 from the 2nd PCR reaction wereligated by 3-fragment ligation into an approx. 3.8-bp-longBgIII/HindIII-pDS56-6HIS-HIV2-gp32 vector fragment. The desired plasmidwas identified by restriction mapping and the PCR-synthesised DNAregions checked by DNA sequencing (construction: pDS56-HIV2-CD44 exon6).

Expression of the HIV2(gp32)-CD44 exon 6 antigen in E. coli

To express the HIV2(gp32)-CD44 exon 6 antigen in E. coli, the E. coliK12 strain RM82 (a methionine revertant of EX8654, Murry, N. E. et al.(1977) Mol. Gen. Genet. 150: 53-61) was transformed with theHIV2(gp32)-CD44 exon 6 expression plasmid pDS56-HIV2-CD44 exon 6(resistance ampicillin) and the IacI repressor plasmid pUHA1 (resistancekanamycin). Production and description of the plasmid pUHA1 aredescribed in Stuber, D. et al. (1990)

Immunol. Methods IV: 121-152.

RM82/pUHA1/pDS56-HIV2-CD44 exon 6 cells were cultured in DYT medium (1%(w/v) yeast extract, 1% (w/v) Bacto Tryptone, Difco, and 0.5% NaCl) with50 mg/l ampicillin and 50 mg/l kanamycin up to an optical density of0.6-0.9 at 550 nm, and then induced with IPTG (1-5 mmol/l endconcentration). After an induction phase of 4-8 h, the cells wereharvested by centrifugation, washed with 10 mmol/l phosphate buffer, pH6.8, and stored at -20° C. until further processing.

The cell pellet from 1 ml of culture medium (RM82/pUHA2/pDS56-HIV2-CD44exon 6 cells) was re-suspended in 0.25 ml 10 mmol/l phosphate buffer, pH6.8, and 1 mmol/l EDTA and the cells mechanically lysed by means of aFrench press. After centrifugation, 1/5 volumes of 5×SDS sample buffer:50 mmol/l Tris-HCl, pH 6.8, 1% SDS, 1% mercaptoethanol, 10% glycerol,and 0.001% bromophenol blue) was added to the supernatant. The insolublecell debris fraction was resuspended in 0.3 ml 1×SDS sample buffer with6-8M urea. The samples were then incubated for 5 min at 95° C. andcentrifuged. Thereafter, the proteins were separated bySDS-polyacrylamide gel electrophoresis (PAGE) (Laemmli, U. K. (1970)Nature 227: 680-685) and stained with Coomassie Brilliant Blue R dye.

The HIV(gp32)-CD44 exon 6 antigen (FIG. 10) synthesized in E. coli washomogeneous and found exclusively in the insoluble cell fraction. Theexpression level for the HIV2(gp32)-CD44 exon 6 antigen was 30-50% inrelation to the E. coli total protein.

Preparation of HIV2(gp32)-CD44 exon 6 antigen from E. coli

Cell lysis and preparation of inclusion bodies (IB's).

20 g (wet weight) of RM82/pUHA1/pDS56-HIV2-CD44 exon 6 cells werere-suspended in 100 ml 0.1 mol/l Tris-HCl, pH 7.0, at 0° C. 30 mglysozyme was added, and the mixture was incubated for 20 min at 0° C.The cells were then lysed completely by mechanical high pressuredispersion, and the DNA was digested in 30 min at 25° C. by addition of2 mmol/l MgCl₂ and 1 mg DNAase (Boehringer Mannheim, Germany, Cat. No.154709). Then 50 ml 60 mmol/l EDTA, 6% Triton X100 and 1.5 mmol/l NaCl,pH 7.0, were added to the digested solution and this mixture incubatedfor a further 30 min at 0° C. The insoluble components (cell debris andIB's) were then centrifuged down on a Sorvall centrifuge. The pellet wasresuspended in 100 ml 0.1 mol/l phosphate buffer, pH 8.5, incubated for30 min at 25° C., and the IB product isolated by centrifugation.

Purification of the HIV2(gp32)-CD44 exon 6 antigen using metal chelatechromatography

The 2.5 g IB pellet (wet weight) was suspended in 25 ml 6 mol/lguanidine-HCl, 0.1 mol/l phosphate buffer, pH 8.5, by stirring for 2 hat 25° C. The insoluble components were separated off by centrifugationand the clear supernatant applied to an NTA column equilibrated with 6mol/l guanidine-HCl , 0.1 mol/l phosphate buffer, pH 8.5 (column volume:50 ml, NTA gel from the Diagen Company, Germany; Hochuli, E. et al.(1988). Bio/Technology 6: 1321-1325).

The column was then washed with about 5 column volumes of 8 mol/l urea,10 mmol/l Tris-HCl, and 0.1 mmol/l phosphate buffer, pH 8.5.Subsequently, the HIV2(gp32)-CD44 exon 6 antigen was eluted with 8 mol/lurea and 0.1 mol/l phosphate buffer, pH 4.0, and the HIV2(gp32)-CD44exon 6 antigen-containing fractions pooled.

Expression and isolation of the HIV2 (gp32)-carrier antigen in E. coli

Analogous to the HIV2 (gp32)-CD44 exon 6 antigen the HIV2 (gp32) carrierantigen was produced by using the plasmid pDS56-6HIS-HIV2-gp32.

Biotinylation of HIV2 (gp32)-CD44 exon 6 antigen

Biotinylation was performed with biotinoyl-★-aminocapronicacid-N-hydroxy- succinimide (Bi-X-NHS) (Boehringer Mannheim, GermanyCat. No. 1003933) in the molar ratios 1:3, 1:6, and 1:10. The fusionprotein HIV2 (gp32)-CD44 exon 6 present in a concentration of 7.1 mg/mlin 8M urea, 0.1M sodium phosphate buffer, 10 mM Tris, pH ca. 6, wasdiluted with dialysis buffer (0.1M sodium phosphate buffer, 0.5% SDS, 10mM DTT, pH 8.5) to 2 mg/ml and dialyzed against the aforementioneddialysis buffer at room temperature and further diluted to 1 mg/ml. Theappropriate quantity of Bi-X-NHS was added to the fusion protein,incubated for 2 h at room temperature, and than the reaction was stoppedby addition of 1M lysine/HCI, pH 6.5, to 2 mM. After dialysis against0.1M sodium phosphate buffer, 0.5% SDS, pH 6.0, the reagent HIV2(gp32)-CD44 exon 6-Bi was stored at -20° C.

The peptide HIV2 (gp32) was biotinylated analogous.

REFERENCES

Hochuli, E.; W. Bannwarth; H. Dbeli; R. Genz; D. Stuber (1988). Geneticapproach to facilitate purification of recombinant proteins with a novalmetal chelate adsorbent. Bio/Technology 6: 1321-1325

Laemmli, U. K. (1970). Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227: 680-685

Mullis, K. B.; F. A. Faloona (1987). Specific synthesis of DNA in vitrovia a polymerase-catalyzed chain reaction. Methods Enzymol. 155: 355-350

Murray, N. E.; W. J. Brammar, K. Murray (1977). Lambdoid phages thatsimplify the recovery of in vitro recombinants. Mol. Gen. Genet. 150:53-61.

Sambrook, J.; E. F. Fritsch, T. Maniatis (1989). Molecular cloning: Alaboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

Screaton, G. R.; M. Bell, D.G: Jackson, F. B: Cornelis, U. Gerth, J. I.Bell (1992). Genomic structure of DNA encoding the lymphocyte homingreceptor CD44 reveals at least alternatively spliced exons. Proc.

Natl. Acad Sci. 89: 12160-12164

Stuber, D.; H. Matile, G. Garotta (1990). System for high levelproduction in Escherichia coli and rapid application to epitope mapping,preparation of antibodies, and structure-function analysis. Inmunol.Methods IV: 121-152

Example 9

Immunization of mice

Immunization of mice was performed according to Cianfriglia et al.(1983). Hybridoma, Vol. 2, No. 4: 451-457. As immunogen syntheticpeptides corresponding to amino acid 1-13, 9-23, 19-33, 29-43 and 1-43coupled to a carrier protein (see example 7) as well as the HIV2(gp32)-CD44 exon 6 antigen were used.

12-week old Balb/c mice were immunized with 50 μg immunogen in completeFreund's adjuvant intraperitoneally 15 and 8 days prior to fusion. 3days before fusion, they were immunized intraperitoneally with 200 μgimmunogen in PBS buffer, and 2 days before fusion and on the last daybefore infusion they were immunized both intraperitoneally andintravenously with 100 μg immunogen in PBS buffer.

Fusion and cloning

Manufacture of spleen cell suspension

Mice were terminated by cervical dislocation, and their spleens removedunder sterile conditions. The spleen cells were teased out of theconnective tissue in RPMI 1640 basic medium. The cell suspension wasthen passed through a sieve and centrifuged at 200 g (centrifuge tubes)in RPMI basic medium.

Fusion

Spleen cells from an immunized mouse were mixed in a ratio 1+5 withP3×63Ag8- 653 myeloma cells (ATCC CRL 8375) and centrifuged (10 min, 300g, 4° C.). The cells were washed again with RPMI basic medium andcentrifuged at 400 g . The supernatant was decanted off and then 1 mlPEG (Mr 4000,Merck) was added and mixed by pipetting. After 1 min on awater bath, 5 ml RPMI 1640 basic medium was added drop-wise at roomtemperature over a period of 5-6 min and the mixture made up to 50 mlwith medium (RPMI 1640+10% FCS). Subsequently this was centrifuged for10 min at 400 g, 4° C. The sedimented cells were added to RPMI 1640medium +10% FCS and seeded in 96-well culture plates at 2.5×10⁴ spleencells per well in 200 μl selection medium (100 μM hypoxanthin, 1 μg/mlazaserin in RPMI 1640+10% FCS) FCS=fetal calf serum!.

After 10 days, these primary cultures were tested for specific antibodysynthesis. Primary cultures of appropriate specificity were cloned in96-well culture plates using an FACS (cell sorter). As growth factor,Interleukin 6 (Boehringer Mannheim Cat. No. 1271172, 100 U/ml) was addedto the medium.

In this way, the following hybridoma cell lines were isolated; they havebeen deposited at the DMS facility in Braunschweig:

MAK<CD44>M-1.1.12

MAK<CD44>M-2.42.3

MAK<CD44>M-4.3.16

For MAK<CD44>M-1.1.12 a synthetic peptide corresponding to amino acids9-23 , for MAK<CD44>M-2.42.3 a synthetic peptide corresponding to aminoacids 29-43 and for MAK<CD44>M-4.3.16 a synthetic peptide correspondingto amino acids 1-13 of the CD44 exon 6 peptide having the amino acidsequence shown in FIG. 7 was used.

Antibody production

Obtaining antibodies from ascites

5×10⁶ hybrid cells were injected i.p. into two mice pre-treated with 0.5ml Pristan. After 1-3 weeks, ascites with an IgG concentration of 5-20mg/ml was obtained. From this antibodies were isolated in the usualmanner.

Obtaining antibodies from cell culture supernatants

Hybridoma cells were multiplied over a period of 7 days at aninoculation density of 1×10⁵ cells/ml in RPMI 1640+10% FCS on a Technebiological stirrer (THERMO-DUX, Wertheim/Main, Model MCS-104XL, Cat. No.144-050). Mean concentrations of 100 μg MAB/ml were achieved in theculture supernatant. Purification was performed using standard proteinchemistry methods.

Example 10

Assessment of the specificity of the produced antibodies

Antibodies to synthetic CD44 peptide

To establish antibody specificity in the hybridoma cell-culturesupernatant, reactivity towards the partial peptide sequence and theentire exon 6 was determined in parallel by inhibition test. 96-welltiter plates (Nunc) were coated with 200 μl/well of streptavidin 10μg/ml, coating buffer=0.2 mol/l sodium carbonate/bicarbonate!. Aftercoating with streptavidin, the biotinylated peptide e.g. 1-13 biotin,9-23 biotin, 19-33 biotin, 29-43 biotin, c=2.5 μg/ml was bound inincubation buffer sodium phosphate buffer, 40 mM, 0.5% Crotein C, 100μl/well, incubation 1 h, room temperature!. The free binding sites weresaturated with blocking buffer 0.9% NaCl, 1% Crotein C, 200 μl, 30 min,room temperature!.

The antibody solution to be tested with and without the free peptideExon 6 (1-43)NH₂, c=5 μg/ml was added and incubated for one hour. Aftera further wash step 0.9% NaCl, 0.05% Tween!, 100 μl of a POD-labelledFab fragment from sheep-sourced polyclonal antibody to mouse-kappa andmouse lambda BM, mouse Ig determination kit, bottle 2 and bottle 6! wasadded. It was incubated for 1 h at room temperature. After a furtherwash step the color substrate, 100 μl, ABTS, BM: #811769, #687359! wasincubated for 30 min at room temperature, The absorbance at 450/490 nmwas measured on a Dynatech MR 700 microplate reader.

All positive antibodies including the deposited cell lines wereafterwards screened by dot-blot and immunohistology.

Antibodies to recombinant CD44 (fusion protein)

To determine antibody specificity from fusions with recombinant CD44 asantigen, additional screening tests were employed. Antibody samples weretested for reactivity with the fusion protein and for cross-reactionwith HIV-gp32 in a parallel ELISA assay. The streptavidin-coatedmicrotiter plates (see section 1) were incubated with biotinylatedfusion protein HIV2(gp32)-CD44 exon 6-Bi(XOSU) or HIV2(gp32)-Bi(XOSU)c=5 μg/ml, 100 μl/well, 1 h room temperature!. The free binding siteswere blocked with blocking buffer 0.9% NaCl, 1% Crotein C, 200 μl, 30min room temperature!. After a wash step 0.9% NaCl, 0.05% Tween! theantibody sample c=5-10 μg/ml, dilted in incubation buffer (40 mM sodiumphosphate buffer!, 100 μl per well and was incubated for 1 h at roomtemperature. The following steps were done as in the above examples forthe synthetic peptide.

Some primary cultures with strong reactivity to the recombinant CD44 andlow cross-reactivity towards HIV2(gp32) protein were obtained. Thesecultures were further assessed by dot-blot and immunohistology.

Determination of specificity of antibodies to cells and tissue(immunostaining)

Method A: Cells from tumor cell lines (e.g. ZR-75 1 or MDA 4A4) weredetached from the flask by scraping and the cell suspensions weredropped onto glass slides, dried and fixed with methanol.

Method B: Freeze-dried sections of tumor and normal tissue were fixedwith acetone.

After blocking with 5% skimmed milk-TBS at 37° C. for 60 min, followedby washing with TBS for 2 min, the sample (undiluted cell-culturesupernatant) was incubated with antibody for 120 min at 37° C. Aftercarefully washing with TBS X3, further incubation was performed withbiotinylated anti-mouse Ig (Dakopatts) for 60 min at 37° C. Afterfurther washing (TBS) HRPO avidin-biotin complex (Dakopatts) was addedand incubated with the sample at room temperature for 60 min. Afterwashing with TBS X1 1% glutaraldehyde solution was added for 1 min atroom temperature. After a further wash step, the substrate (DAB) wasadded and incubated with the sample (15-20 min). After washing with tapwater the nuclei were stained with hematoxylin for 30 sec. The sampleswere dried and embeded with Cristal Mount (Kaiser's jelly).

The results obtained with monoclonal antibody from cell linesMAB<CD44>M-1.1.12 and 4.3.16 are presented in Table 4. In Method B, theMAB 1.1.12 shows high specificity for tumor tissue from the lung, colonand bladder and MAB 4.3.16 revealed specificity for tumor tissue fromthe colon. In Method A, MAB 1.1.12 and MAB 4.3.16 showed increasedreactivity to the cell line ZR-75-1 (exon 6 high-producer), a humanbreast cancer cell line (ATCC CRL 1500) than to the cell line MDA4A4(exon 6 low-producer). This cell line is a subclone of cell lineMDA-MB-435S (ductal carcinoma, breast, human; ATCC HTB 129; the subclonewas produced according to Bao et al, Differentiation 52 (1993),239-246;MDA4A4 is identical to MDA-MB-435-C2 of this reference).

Within the primary cultures obtained with the recombinantly producedCD44 fusion protein as immunogen (see above) the culture PK 9.00.22showed a high specificity to tumor tissue of colon with method B. Withmethod A this cultured cell line showed also a marked specificity forthe cell line ZR 75-1.

                  TABLE 4    ______________________________________    Results of Immunostaining    Method A    cell suspension    ZR75-1        MDA4A4            Method B    exon 6 high-  exon 6 low-       tissue    producer      producer          tumor normal    ______________________________________    MAK 1.1.12            +         -         lung  +     -                                colon +     -                                bladder                                      +     -    MAK 4.3.16            +         -         colon +     -    ______________________________________     + strong reaction;     - weak reaction

Determination of specificity of produced antibodies by dot-blot

Preparation of cell extracts

Cells of lines HT29 (ATCC HTB 38--colon adenocarcinoma) and MDA4A4 werecultured in a medium according to ATCC catalogue and were harvested withor without protease additive.

The cells harvested without protease additive were centrifuged, added todouble the volume of lysis buffer (50 mM potassium phosphate buffer, 150mM NaCl, pH 8.0), homogenised for 5 min in a Dounce homogenizer and thequantity of protein determined. On the basis of this protein value, thecellular suspension was adjusted to a protein concentration of 1-2 mg/mlusing lysis buffer with or without detergent 1% Triton X-100 (BoehringerMannheim, Germany Cat. No. 743119), 0.6% CHAPS (Boehringer Mannheim,Germany Cat. No. 810681), 1% HECAMEG (Boehringer Mannheim, Germany Cat.No. 1382225), 0.9% octyl glucoside (Boehringer Mannheim, Germany Cat.No. 411469) or 0.05% dodecylmaltoside (Boehringer Mannheim, Germany Cat.No. 808342)! and stirred for 2 h. After the centrifugation, thesupernatant which contains CD44 or CD44v was stored at 4° C. or -20° C.,and use unchanged. The supernatant obtained after centrifuging off themembranes contained sufficient CD44 (standard form) and CD44v (CD44 withadditional exons) for antibody assessment. Because of the mRNAconcentration in the cells, it is assumed that MDA4A4 containspredominantly CD44-standard form and hardly any exon 6- containingCD44v. HT29 cells, on the other hand, should contain mainly exon 6-containing CD44v.

A further simple way in which CD44 or CD44v can be obtained is toharvest the cells with trypsin instead of the aforementioned cellharvest with subsequent cellular separation. The supernatant obtainedafter addition of trypsin inhibitor and centrifuging off the cells alsocontains sufficient CD44 and CD44v for antibody assessment.

Assessment of antibodies by dot blot

Various solutions (synthetical produced CD44 exon6 peptide with theamino acid sequence 1-43 as shown in FIG. 7 according to example 7, HT29cellular extract, MDA4A4 cellular extract) were applied tonitrocellulose by capillary tubes. After blocking with Crotein C,incubation of the nitrocellulose with the antibodies (AB) took place. Asantibodies the supernatant of the various MAB<CD44>-M cell lines wasused. Detection of bound Ab is done with a polyclonal anti Ig antibodyconjugated to alcalic phosphatase. For color reaction NBT/X phosphatewas used.

The specificity of the reaction can be shown by addition of free Exon6peptide to the Ab before incubation of the nitrocellulose. If thereaction is specific for Exon6 or CD44v, either no or only very slightbinding of the AB to the nitrocellulose takes place after addition ofthe free peptide. Best results were obtained with the following clones:

MAB<CD44>M-1.1.12

MAB<CD44>M-2.42.3

The following compounds were spotted onto the nitrocellulose (Schleicher& Schuell 401180) using capillary tubes:

A: synthetically produced CD44 exon 6, 1-43-NH₂, (0.1 mg/ml)

B: HT29 extract (1.2 mg/ml)

C: MDA4A4 extract (1.35 mg/ml)

After blocking the nitrocellulose with incubation buffer (20 mMTris/HCl, 150 mM NaCl, 1% Crotein C, pH 7.4), in each case one blot with2 or 3 dots was incubated using cell culture supernatant (in each caseundiluted or diluted 1:4, 1:16, 1:64, 1:256 and 1:1024 in incubationbuffer). The bound antibody was detected with PAB<M-Ig>S-Fab-AP and5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue-tetrazolium chloride(NBT/X phosphate) as color substrate.

To test the AB specificity the test was run twice in parallel,performing pre-incubation of the antibody in one of the tests using 10fg/ml of the free Exon6, 1-43 peptide. Inhibition should be seen for anexon 6-specific reaction.

The antibodies produced by the cell lines MAK<CD44>M-1.1.12 andMAK<CD44>M-2.42.3 are able to bind to dotted CD44 exon 6 peptide and toan extract of HT29 cells but not to an extract of MDA4A4 cells. Bindingof the monoclonal antibody to dotted CD44 exon 6 peptide and to anextract of HT29 cells is specific for the tumorspecific variant of CD44vbecause preincubation of both antibodies with synthetic CD44 exon 6inhibits the binding of the antibodies to nitrocellulose (Table 5).

                                      TABLE 5    __________________________________________________________________________    Results of dot-blots               Binding to extract of                              Binding to extract of    Dot-Blot results               HT 29-cells    MDA4A4-cells    __________________________________________________________________________    monoclonal antibody               no preincubation                       preincubation                              no preincubation                                      preincubation               of mAb  of mAb with                              of mAb  of mAb with                       peptide exon 6 peptide exon 6    MAK<CD44>M-1.1.12               moderate                       low (weak                              no      no                       inhibition)    MAK<CD44>M-2.42.3               strong  no (strong                              no      no                       inhibition)    __________________________________________________________________________

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 2    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 141 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 7..135    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    GCTACCACTTTGATGAGCACTAGTGCTACAGCAACTGAGACAGCAACC48    ThrLeuMetSerThrSerAlaThrAlaThrGluThrAlaThr    1510    AAGAGGCAAGAAACCTGGGATTGGTTTTCATGGTTGTTTCTACCATCA96    LysArgGlnGluThrTrpAspTrpPheSerTrpLeuPheLeuProSer    15202530    GAGTCAAAGAATCATCTTCACACAACAACACAAATGGCTGGTACG141    GluSerLysAsnHisLeuHisThrThrThrGlnMetAla    3540    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    ThrLeuMetSerThrSerAlaThrAlaThrGluThrAlaThrLysArg    151015    GlnGluThrTrpAspTrpPheSerTrpLeuPheLeuProSerGluSer    202530    LysAsnHisLeuHisThrThrThrGlnMetAla    3540    __________________________________________________________________________

We claim:
 1. Antibody that specifically binds to the peptidecorresponding to CD44 exon 6 having the amino acid sequence shown inFIG. 7 (SEQ ID NO:1), its allele variations and phosphorylation andglycosylation products.
 2. Antibody according to claim 1 which ismonoclonal or polyclonal.
 3. Monoclonal antibody which specificallybinds to the peptide corresponding to CD44 exon 6 having the amino acidsequence shown in FIG. 7 (SEQ ID NO:1) obtainable by the hybridoma celllines MAK<CD44>M-1.1.12, MAK<CD44>M-2.42.3 or MAK<CD44>M-4.3.16,Accession Numbers DSM ACC2156, DSM ACC2157 or DSM ACC2158, respectively.4. Monoclonal antibody according to claim 1 which recognize the sameepitope as the monoclonal antibodies produced by the hybridoma celllines MAK<CD44>M-1.1.12, MAK<CD44>M-2.42.3 or MAK<CD44>M-4.3.16,Accession Numbers DSM ACC2156, DSM ACC2157 or DSM ACC2158, respectively.5. Method for the production of an antibody according to claim 1comprising:injecting a suitable laboratory animal with an effectiveamount of an antigenic compound comprising the peptide corresponding toCD44 exon 6 having the amino acid sequence shown in FIG. 7 (SEQ IDNO:1), or its allele variations, collecting serum from this animal, andisolating the specific antibody by immunoabsorbant techniques.
 6. Methodfor the production of an antibody according to claim 1comprising:injecting a suitable laboratory animal with an effectiveamount of an antigenic compound comprising the peptide corresponding toCD44 exon 6 having the amino acid sequence shown in FIG. 7 (SEQ IDNO:1), or its allele variations, isolating the antibody producing cells,immobilizing these cells, screening for the immortal cell line producingthe antibody according to claim 1, cloning said immortal said cell line,and obtaining the antibody from ascites or the supernatant of thecultured immortal cell line.
 7. Method for the production of an antibodyaccording to claim 5, wherein as immunogen a fusion protein comprisingthe peptide corresponding to CD44 exon 6 having the amino acid shown inFIG. 7 (SEQ ID NO:1), or its allele variations or a peptide of at leastsix amino acid length corresponding to an amino acid sequence of CD44exon 6 shown in FIG. 7, or its allele variations which is coupled to asuitable immunogenic carrier is used.
 8. Immunoassay for the detectionof a CD44 protein contained in the peptide corresponding to CD44 exon 6wherein an antibody that specifically binds to the peptide CD44 exon 6having the amino acid sequence shown in FIG. 7 (SEQ ID NO:1), or itsallele variations is used.
 9. Test kit comprising at least one antibodyaccording to claim
 2. 10. Monoclonal antibody according to claim 1,which is an antigen-binding fragment, or a humanized or human antibody.11. Monoclonal antibody according to claim 10, which is a human IgG1antibody.
 12. Immunoassay according to claim 8 for cancer diagnosis. 13.Immunoassay according to claim 8, for the detection of a complex of theantigen with a second antibody.